Decoy probes

ABSTRACT

The present invention features inhibitors of target-independent amplification and the use of such inhibitors for enhancing an amplification protocol. The inhibitors are believed to enhance an amplification protocol by inhibiting the ability of one or more nucleic acid polymerases to use nucleic acid in a polymerase reaction in the absence of target nucleic acid.

This application is a continuation of application Ser. No. 09/365,121,filed Jul. 30, 1999, now U.S. Pat. No. 6,297,365, the contents of whichare hereby incorporated by reference herein, which claims the benefit ofU.S. Provisional Application No. 60/094,979, filed Jul. 31, 1998.

FIELD OF THE INVENTION

The present invention features compositions, reagents and methods forenhancing an amplification protocol. Preferably, the compositions,reagents and methods are used in conjunction with an RNA polymerasedriven transcription-associated amplification protocol.

BACKGROUND OF THE INVENTION

None of the references described herein are admitted to be prior art tothe claimed invention.

Nucleic acid amplification involves the enzymatic synthesis of nucleicacid amplicons that contain a sequence complementary to a nucleic acidsequence being amplified. Nucleic acid amplification can be performedusing different techniques such as those involvingtranscription-associated amplification, the polymerase chain reaction(PCR), ligase chain reaction (LCR) and strand displacement amplification(SDA).

Uses of nucleic acid amplification include diagnostic and syntheticapplications. Diagnostic applications of nucleic acid amplificationtypically involve screening for whether amplicons are produced, theamount of amplicon produced, and/or determining whether producedamplicons contain a particular sequence.

Transcription-associated amplification of a nucleic acid sequencegenerally employs an RNA polymerase, a DNA polymerase,deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and apromoter-template complementary oligonucleotide. The promoter-templatecomplementary oligonucleotide contains a 5′ sequence recognized by anRNA polymerase and a 3′ sequence that hybridizes to a template nucleicacid in a location 3′ of a target sequence that is sought to beamplified. After hybridization of the promoter-template complementaryoligonucleotide to the template, a double-stranded promoter is formedupstream from the target sequence. Double-stranded promoter formationgenerally involves DNA polymerase activity.

RNA polymerase-associated amplification is initiated by the binding ofan RNA polymerase to a promoter region that is usually double-stranded.The RNA polymerase proceeds downstream from the promoter region andsynthesizes ribonucleic acid in a 5′ to 3′ direction. Multiple copies,generally in the range of 100-3,000 RNA transcripts, can be produced byRNA polymerase-associated amplification using a single template.

Different formats can be employed for performingtranscription-associated amplification. Examples of different formatsare provided in publications such as Burg et al., U.S. Pat. No.5,437,990; Kacian et al., U.S. Pat. No. 5,399,491; Kacian et al., U.S.Pat. No. 5,554,516; Kacian et al., International Application No.PCT/US93/04015, International Publication No. WO 93/22461; Gingeras etal., International Application No. PCT/US87/01966, InternationalPublication No. WO 88/01302; Gingeras et al., International ApplicationNo. PCT/US88/02108, International Publication No. WO 88/10315; Davey andMalek, European Application No. 88113948.9, European Publication No. 0329 822 A2; Malek et al., U.S. Pat. No. 5,130,238; Urdea, InternationalApplication No. PCT/US91/00213, International Publication No. WO91/10746; McDonough et al., International Application No.PCT/US93/07138, International Publication No. WO 94/03472; and Ryder etal., International Application No. PCT/US94/08307, InternationalPublication No. WO 95/03430. (Each of these references is herebyincorporated by reference herein.)

PCR amplification is described by Mullis et al., U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159, and in Methods in Enzymology,155:335-350 (1987). (Each of these references is hereby incorporated byreference herein.)

An example of LCR is described in European Patent Publication No. 320308 (hereby incorporated by reference herein). LCR uses at least fourseparate oligonucleotides. Two of the oligonucleotides hybridize to anucleic acid template so that the 3′ end of one oligonucleotide and the5′ end of the other oligonucleotide are positioned for ligation. Thehybridized oligonucleotides are then ligated forming a full-lengthcomplement to the target sequence in the nucleic acid template. Thedouble-stranded nucleic acid is then denatured, and third and fourtholigonucleotides are hybridized to the complementary strand and joinedtogether. Amplification is achieved by further cycles of hybridization,ligation, and denaturation, producing multiple copies of the targetsequence and the sequence complementary to the target sequence.

SDA is an isothermal amplification reaction based on the ability of arestriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its recognition site, and on the ability ofa DNA polymerase to initiate replication at the nick and displace adownstream non-template strand. (See, e.g., Walker, PCR Methods andApplications, 3:25-30 (1993), Walker et al., Nucleic Acids Res.,20:1691-1996 (1992), and Walker et al., Proc. Natl. Acad. Sci.,89:392-396 (1991). Each of these references is hereby incorporated byreference herein.) The steps used in generating fragments for carryingout autocatalytic SDA amplification are indicated to be adaptable forgenerating fragments for transcription-associated amplification oramplification carried out using Q-beta technology. (Walker et al.,Nucleic Acids Res., 20:1691-1696 (1992).)

SUMMARY OF THE INVENTION

The present invention features inhibitors of target-independentamplification and the use of such inhibitors for enhancing anamplification protocol. The inhibitors are believed to enhance anamplification protocol by inhibiting the ability of one or more nucleicacid polymerases to use nucleic acid in a polymerase reaction in theabsence of target nucleic acid.

“Target-independent amplification” refers to the amplification of anucleic acid sequence that is not a target nucleic acid sequence. Thetarget nucleic acid sequence is present on a target nucleic acid and isa nucleotide base sequence, or region, sought to be amplified.

It is believed that the present invention benefits nucleic acidamplification by using competitors of amplification oligonucleotides tosequester amplification enzymes in solution from non-target nucleic acidsuch as amplification oligonucleotides. The competitors appear tocompete with amplification oligonucleotides for binding to one or moreamplification enzymes, and may be added in an excess amount relative tothe amplification oligonucleotides.

In the absence of target nucleic acid, affected amplification enzymesare occupied by the competitors, and the ability of the enzymes toparticipate in a polymerase reaction involving non-target nucleic acidis inhibited. Amplification oligonucleotides hybridized to targetnucleic acid favorably compete with the competitors for amplificationenzyme binding. Thus, the competitors function as reversible inhibitorsof amplification enzymes.

Amplification enzymes are nucleic acid polymerases that catalyze thesynthesis of polynucleotides by polymerizing nucleoside triphosphates.Reversible inhibition of amplification enzymes is carried out to preventthe formation of undesirable side-products, such as one or more of thefollowing: (1) a primer-dimer; (2) an RNA replicating nucleic acid; (3)a single-stranded primer extended RNA or DNA; and (4) a modification toa primer rendering the primer unable to participate in the amplificationof a target nucleic acid sequence. The types of undesirableside-products that can be formed will depend upon the particularamplification protocol that is performed.

Amplification oligonucleotides hybridize to target nucleic acid andparticipate in an amplification reaction. Examples of amplificationoligonucleotides include template-complementary probes, such as primers,and promoter-template complementary probes, such as promoter-primers.

While inhibitors of target-independent amplification described by thepresent invention are expected to function by competing withamplification oligonucleotides for binding to an amplification enzyme,unless otherwise specified in the claims, the claims are not limited toa particular mechanism. For example, probes having a high degree ofsequence similarity to an RNA polymerase promoter which enhance anamplification protocol are described in the examples provided below.Such examples illustrate the effectiveness of such probes and allow forprobe design based on sequence similarity to an RNA polymerase promoterwithout determining enzyme binding or the ability to compete withamplification oligonucleotides.

Thus, a first aspect of the present invention describes a decoy probecomprising,

a first nucleotide base recognition sequence region, wherein the firstregion binds to an RNA polymerase, and

an optionally present second nucleotide base recognition sequenceregion,

provided that if the first region is nucleic acid, then the secondregion is either directly joined to the 5′ end of the first region or isjoined to the 3′ end or 5′ end of the first region by a non-nucleotidelinker,

wherein the optionally present second region is present if the firstregion can be used to produce a functional double-stranded promotersequence using a complementary oligonucleotide,

further provided that if the first region is nucleic acid which can beused to produce the functional double-stranded promoter sequence usingthe complementary oligonucleotide, then the decoy probe does not have anucleic acid sequence greater than about 10 nucleotides in length joineddirectly to the 3′ end of the first region.

The first region contains a nucleotide base recognition sequence regionto which an RNA polymerase can bind. An example of such a sequence isone sense of a double-stranded promoter sequence. The first region cancontain, for example, a derivative of one sense of a promoter region,where the derivative cannot be used to produce a functionaldouble-stranded promoter sequence when made double-stranded.

If the decoy probe can form a functional promoter, then it is desirablenot to have significant downstream sequences that can be used as anamplifiable template. Preferably, if the first region can be used toproduce a functional double-stranded promoter then the decoy probe doesnot have a nucleotide base sequence greater than 5 nucleotides in lengthjoined directly to the 3′ end of the first region.

The presence of a second region positioned 3′ or 5′ to the first regiondoes not prevent other regions from being present. For example, thedecoy probe may contain a second region 3′ to the first region and mayalso contain a region joined either directly, or through anon-nucleotide linker, to the 5′ end of the first region.

Preferably, the decoy probe is a purified probe. By “purified” is meantthat the decoy probe makes up at least 0.1% of the recognition moleculespresent in a preparation. In preferred embodiments, the decoy probemakes up at least 1%, at least 5%, at least 25%, at least 50%, at least75%, or 100% of the nucleic acid present in a preparation.

A “nucleotide base sequence recognition molecule” is a moleculecontaining nucleotide base recognition groups linked together by abackbone. Examples of nucleotide base sequence recognition moleculesinclude peptide nucleic acids, oligonucleotides, and derivativesthereof. A nucleotide base recognition group can hydrogen bond toadenine, guanine, cytosine, thymine or uracil. The backbone presents thenucleotide base recognition groups in a proper conformation for hydrogenbonding to a complementary nucleotide present in a nucleic acidsequence.

A “functional double-stranded promoter sequence” is a sequence that isrecognized by an RNA polymerase and can be used to produce readilydetectable RNA transcripts. A functional double-stranded promoter can beformed from a single-stranded promoter sequence, for example, byhybridizing to the promoter sequence a complementary oligonucleotide.

A “non-nucleotide linker” refers to one or more chemical moieties whichform a stable linkage under amplification conditions and which do notcontain a nucleotide base recognition group that can act as a templatein a polymerase reaction.

Another aspect of the present invention describes a decoy probecomprising,

a first nucleotide base recognition sequence region, wherein the firstregion has at least 35% sequence similarity to an RNA polymerasepromoter sequence, and

an optionally present second nucleotide base recognition sequenceregion,

provided that if the first region is nucleic acid, then the secondregion is either directly joined to the 5′ end of the first region or isjoined to the 3′ end or 5′ end of the first region by a non-nucleotidelinker,

wherein the optionally present second region is present if the firstregion can be used to produce a functional double-stranded promotersequence using a complementary oligonucleotide,

further provided that if the first region is nucleic acid which can beused to produce the functional double-stranded promoter sequence usingthe complementary oligonucleotide, then the decoy probe does not have anucleic acid sequence greater than about 10 nucleotides in length joineddirectly to the 3′ end of the first region.

Decoy probe binding to an RNA polymerase can be measured using standardtechniques, such as through the use of competitive and noncompetitiveassays employing a labeled oligonucleotide having an RNA polymerasepromoter sequence. Additionally, oligonucleotides binding to RNApolymerase can be selected for and produced in large quantities usingthe “Protein Binding Amplification Protocol” described infra.

Another aspect of the present invention describes a reagent mixture foruse in an amplification reaction. The mixture contains a nucleic acidpolymerase and a reversible inhibitor of the polymerase. The mixturedoes not contain a nucleic acid substantially complementary to theinhibitor. Thus, the mixture does not contain a nucleic acid that wouldhybridize to the inhibitor under the amplification conditions in whichthe mixture is employed.

The reagent mixture is particularly useful for providing an opportunityfor the reversible inhibitor to bind with the amplification nucleic acidpolymerase prior to exposure to amplification oligonucleotides.Preferably, the mixture does not contain the target sequence to beamplified.

Another aspect of the present invention describes an amplificationprocedure for amplifying a target nucleic acid sequence comprising thesteps of:

a) producing a mixture comprising an amplification enzyme and areversible inhibitor of the enzyme, where the reversible inhibitor doesnot hybridize to a target nucleic acid comprising the target nucleicacid sequence under amplification conditions and the mixture does notcontain the target nucleic acid,

b) providing the mixture to the target nucleic acid, and

c) amplifying the target nucleic acid sequence under the amplificationconditions.

Another aspect of the present invention describes atranscription-associated amplification procedure comprising the step ofamplifying a nucleic acid sequence to produce multiple copies of RNAtranscripts by combining together, under transcription-associatedamplification conditions, a mixture comprising a target nucleic acidcomprising the target nucleic acid sequence, a promoter-templatecomplementary probe, a DNA polymerase, an RNA polymerase, ribonucleosidetriphosphates, deoxyribonucleoside triphosphates, and means forreversibly inhibiting the RNA polymerase. The means for reversiblyinhibiting the RNA polymerase does not hybridize to the target nucleicacid under the amplification conditions to form a stableinhibitor:target complex.

“Means for reversibly inhibiting” refers to material described in thepresent application and equivalents thereof that can reversibly inhibitthe activity of an amplification enzyme.

Another aspect of the present invention describes an improved method ofamplifying a target nucleic acid sequence. The improvement comprises thestep of providing a nucleic acid polymerase used in the amplificationwith means for reversibly inhibiting the polymerase prior to providingthe polymerase to a target nucleic acid comprising the target nucleicacid sequence.

Expected advantages of the present invention include one or more of thefollowing:

(1) increased yield of target complementary amplicons; (2) increasedsensitivity; and (3) increased availability of polymerases for targetamplification. Such advantages are expected to arise from the reductionof undesirable side-products.

Another advantage of the present invention is that it can be employed atan essentially constant temperature. At an essentially constanttemperature, a reaction is not cycled between a high and a lowtemperature to alternatively denature and anneal nucleic acid, such asthat occurring in PCR.

Various examples are used throughout the application. These examples arenot intended in any way to limit the claimed invention.

Other features and advantages of the invention will be apparent from thefollowing figures, detailed description of the invention, examples, andthe claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate a possible mechanism of competition betweendecoy probes and unbound amplification primers for amplification enzymesin a transcription-associated amplification. FIG. 1A illustrates anamplification scheme in the absence of decoy probes. FIG. 1B illustratesdecoy probes inhibiting the formation of side-products.

FIGS. 2A and 2B illustrate two examples of decoy probes. The decoy probeshown in FIG. 2A is a single-stranded oligonucleotide having aself-complementary 5′ end that forms a hairpin. The decoy probe shown inFIG. 2B contains two oligonucleotide regions linked together at their 5′ends to form a decoy probe having two 3′ ends. The three-carbon groups(-ccc) illustrated in FIGS. 2A and 2B are propyl blocking groupsattached to the 3′ ends.

FIG. 3 illustrates the results of an experiment examining the affect ofa blocked decoy probe of SEQ. ID. NO: 10 on T7 hepatitis C virus (HCV)transcription-associated amplification kinetics. Amplification timerefers to the length of time following enzyme reagent addition.Reactions (20 μL reaction volume) were terminated by the addition of HPAprobe reagent. Results with no decoy probes present are indicated by“-♦-”. Results with decoy probes present are indicated by “-▪-”.

FIG. 4 illustrates the results of an experiment examining the affect ofa blocked decoy probe of SEQ. ID. NO: 10 on T7 Human ImmunodeficiencyVirus (HIV) transcription-associated amplification kinetics.Amplification time refers to the length of time following enzyme reagentaddition. Reactions were terminated by the addition of HPA probereagent. Results with no decoy probes present are indicated by “-♦-”.Results with decoy probes present are indicated by “-▪-”.

FIG. 5 illustrates the results of an experiment examining the affect ofa blocked decoy probe of SEQ. ID. NO: 10 on T3 HIVtranscription-associated amplification kinetics. Amplification timerefers to the length of time following enzyme reagent addition.Reactions were terminated by the addition of HPA probe reagent. Resultswith no decoy probe present are indicated by “-♦-”. Results with decoyprobe present are indicated by “-▪-”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features compositions, reagents and methods forenhancing an amplification protocol. The present invention is believedto enhance amplification by providing a means of sequesteringamplification enzymes from amplification oligonucleotides not hybridizedto a target nucleic acid.

FIGS. 1A and 1B illustrate a possible mechanism by which amplificationis enhanced through the use of reversible inhibitors (e.g., decoyprobes) of amplification enzymes. The illustrated mechanism involvestranscription-associated amplification and shows an inhibition of theability of reverse transcription and RNA polymerase to produceundesirable products.

The top of FIG. 1 illustrates amplification in the absence of reversibleamplification enzyme inhibitors (e.g., decoys) and the bottom of FIG. 1illustrates amplification in the presence of reversible amplificationenzyme inhibitors (e.g., decoys). Primers not bound to target nucleicacid are labeled in the figure as “Free Primers”. It is believed thatdecoy probes bind to the amplification enzymes, thereby occupying theenzymes and reducing the ability of the enzymes to form undesirableproducts using amplification oligonucleotides.

The present invention is preferably used to increase the number oftarget specific amplicon products, and to minimize undesirable productsthat unproductively consume reactants and system resources. Bymaximizing the amount of target specific amplicon product and reducingundesirable products, one or more features of amplification can beimproved.

I. Definitions

Descriptions, along with preferred embodiments of some of the termsdescribed herein, are presented in this section. This section is notintended to provide a description of all of the terms used herein, butrather provides a reference section for several of the terms.

“Amplification conditions” refer to conditions compatible with nucleicacid polymerization to produce a complementary strand using a nucleicacid template and a nucleic acid polymerase. Such conditions include thepresence of required amplification components including enzymes,nucleoside triphosphate substrates, buffer conditions, and appropriatetemperature. The specific conditions employed depend upon the type ofamplification being performed. Conditions for performing different typesof amplification are well known in art and are exemplified by thepublications cited herein, such as those discussed in the “BACKGROUND OFTHE INVENTION” supra. Examples of different amplification proceduresexemplified in the “BACKGROUND OF THE INVENTION” includetranscription-associated amplification, PCR, LCR and SDA.

Transcription-associated amplification conditions are those conditionscompatible with RNA polymerase associated amplification involving theproduction of RNA transcripts. Such conditions are well known in the artand include the appropriate buffer conditions, nucleoside triphosphatesubstrates, temperature, amplification oligonucleotides, RNA polymerase,and reverse transcriptase.

SDA conditions are those conditions compatible with strand displacementamplification. Such conditions are well known in the art and include theappropriate buffer conditions, nucleoside triphosphate substrates,temperature, amplification oligonucleotides, nucleic acid polymerase,and restriction enzyme.

DNA polymerase amplification conditions are conditions compatible withDNA polymerase activity. Such conditions include the appropriate bufferconditions, nucleoside triphosphate substrates, and temperature.

An “amplification oligonucleotide” refers to an optionally modifiedoligonucleotide able to participate in an amplification reaction. Thecomposition of an amplification oligonucleotide will depend upon theamplification scheme employed. Examples of amplificationoligonucleotides include primers and promoter-primers which may becomplementary to an initial template, or to a complementary templateproduced from the initial template.

An “analogous oligonucleotide” refers to an optionally modifiedoligonucleotide that has substantially the same nucleic acid sequence asthat present on a target nucleic acid in a region 5′ of the targetsequence. The analogous oligonucleotide has sufficient complementarityto hybridize to the complement of the target nucleic acid underamplification conditions. The analogous oligonucleotide may contain anon-complementary region, such as a promoter region. The analogousoligonucleotide may contain one or more modifications, such as amodification inhibiting nucleic acid polymerase activity. Preferably,the analogous oligonucleotide contains at least about 15 contiguousbases that are at least 80%, more of preferably at least 90%, and mostpreferably 100% analogous to a contiguous base region the target nucleicacid. The analogous oligonucleotide is preferably 15 to 60 optionallymodified nucleotides in length, and more preferably the optionallymodified nucleotides are unmodified nucleotides.

An “analogous primer” refers to an analogous oligonucleotide thatcontains a 3′ end which can participate in a nucleic acid polymerasereaction. The 5′ region of the analogous primer can be non-complementaryto the target nucleic acid, and can be, for example, a promoter sequencethat would result in an analogous promoter-primer.

A “template-complementary oligonucleotide” refers to an optionallymodified oligonucleotide sufficiently complementary to hybridize to atarget nucleic acid in a region 3′ of the target sequence. Thetemplate-complementary oligonucleotide may contain a non-complementaryregion such as a 5′ promoter-region. Preferably, thetarget-complementary oligonucleotide contains at least about 15contiguous bases that are at least 80%, more preferably at least 90% andmost preferably 100% complementary to a contiguous base region of thetarget nucleic acid. The template-complementary oligonucleotide ispreferably 15 to 60 optionally modified nucleotides in length, and morepreferably, the optionally modified nucleotides are unmodifiednucleotides.

A “template-complementary primer” refers to a template-complementaryoligonucleotide that contains a 3′ end that can be readily used in apolymerase reaction. The 5′ region of the primer can benon-complementary to the target nucleic acid, and can be, for example, apromoter sequence.

A “promoter-template complementary oligonucleotide” refers to atemplate-complementary oligonucleotide having a 5′ promoter sequence.The promoter sequence is recognized by an RNA polymerase.

A “primer” refers to an oligonucleotide that contains a 3′ end that canbe readily used in a polymerase reaction. The 5′ region of the primercan be non-complementary to the target nucleic acid, and can be, forexample, a promoter sequence.

A “promoter-primer” refers to an oligonucleotide having a 5′ promotersequence and a 3′ primer sequence.

II. Reversible Inhibition of Amplification Enzyme Activity

Reversible inhibition of amplification enzyme activity is performed toinhibit target-independent amplification or nucleic acid polymeraseactivity. Preferably, the inhibitor used to achieve reversibleinhibition is designed to inhibit the binding of an amplificationoligonucleotide by an amplification enzyme in the absence of a targetnucleic acid. In the presence of the target, the amplificationoligonucleotide forms a complex with the target that effectivelycompetes with the inhibitor for enzyme binding.

Binding of an inhibitor to an amplification enzyme, as opposed tobinding of an amplification enzyme to an amplification oligonucleotide,can be enhanced by techniques such as: (1) increasing the amount of theinhibitor relative to the amplification oligonucleotide; and (2)combining the amplification enzyme with the inhibitor prior to theintroduction of the amplification oligonucleotides.

III. Decoy Probes

Preferably, the methods described herein are performed using a decoyprobe. Preferred decoy probes do not have a 3′ end that can be used in apolymerase reaction. More preferably, the decoy probe does not contain asequence substantially complementary to a target nucleic acid sequence.Substantially complementary sequences can hybridize together underconditions used in an amplification reaction. Preferably, the decoyprobe contains no more than 5 recognition groups able to hydrogen bondwith a region of 10 or more contiguous target nucleic acid sequencebases.

More preferably, decoy probes are used in an amount equal to, or inexcess of, the amount of amplification oligonucleotides. A preferredmolar ratio of decoy probe to total amount of amplificationoligonucleotide is 1:3 to 100:1, preferably 2:1 to 5:1. Preferably, thedecoy probe to amplification enzyme molar ratio is about 1:15 to 5:1,more preferably 1:2 to 2:1, and more preferably 1:1.

Preferably, the decoy probes are incubated with amplification enzyme(s)prior to the introduction of amplification oligonucleotides. Morepreferably, the amplification enzyme(s) are an RNA polymerase and/or areverse transcriptase which are combined with decoy probes having a 5′promoter-like sequence and a blocked 3′ end, prior to contact with anamplification oligonucleotide.

Decoy probes are preferably designed so as not to be used in a nucleicacid polymerase reaction. Preferred decoy probes do not have a structurethat provides a functional promoter and do not have a 3′ end that canparticipate in a polymerase reaction.

Single-stranded and double-stranded DNA promoter sequences that can beefficiently used by an RNA polymerase are preferably avoided. The use ofdecoy probes having a functional double-stranded DNA promoter sequencemight result in the production of undesirable target-independentamplification products.

Similarly, RNA having a stem loop secondary structure and DNA having acircular structure which can result in target-independent amplificationare preferably avoided. Biebricher and Luse, EMBO J., 13:3458-3465(1996), indicates that structured RNA molecules of different sequencescan participate in RNA polymerase catalyzed replication. Daubendiek etal., J. Am. Chem. Soc., 117:7818-7819 (1995), indicates that a circulardeoxyoligonucleotide can serve as a template for T7 polymerase in theabsence of RNA primers, in the absence of RNA promoter sequences, and inthe absence of any duplex structure at all.

A. Decoy Probe Targeting

Preferred decoy probes described by the present invention are targetedto a nucleic acid polymerase, preferably RNA polymerase and/or reversetranscriptase. Such probes can be produced, for example, by employingone or more of the following procedures: (1) selecting for probes whichbind to a RNA polymerase and/or reverse transcriptase; (2) designingprobes having a promoter sequence similar or identical to an RNApolymerase promoter sequence; and (3) designing probes having aself-complementary “hairpin” structure.

RNA promoter sequences from a variety of different sources have beensequenced. (See, e.g., Jorgensen et al., J.B.C., 266:645-655 (1991),hereby incorporated by reference herein.) Examples of naturallyoccurring RNA promoter sequences are as follows:

T3 (+)  SEQ. ID. NO: 1: taatattaac cctcactaaa gggaga; T3 (−)  SEQ. ID.NO: 2: tctcccttta gtgagggtta atatta; T7 (+)  SEQ. ID. NO: 3: taatacgactcactataggg aga; T7 (−)  SEQ. ID. NO: 4: tctccctata gtgagtcgta tta;SP6 (+) SEQ. ID. NO: 5: atttaggtga cactatagaa gag; and SP6 (−) SEQ. ID.NO: 6: ctcttctata gtgtcaccta aat.

A decoy probe can contain a sequence similar to either sense of apromoter. Reference to “similar” also includes the same sequence.Preferably, the decoy probe used in a transcription-associatedamplification reaction has a sequence which is similar to the promotersequence present in a promoter-primer used in thetranscription-associated amplification reaction.

Preferably, decoy probes have a region with a sequence similarity thatis at least 50% similar to the T7 RNA polymerase promoter sequence, theT3 RNA polymerase promoter sequence, or the SP6 RNA polymerase promotersequence. More preferably, sequence similarity is at least 75% similarto the T7 RNA polymerase promoter sequence, the T3 RNA polymerasepromoter sequence, or the SP6 RNA polymerase promoter sequence. Evenmore preferably, sequence similarity is 75% to 95% similar to the T7,T3, or SP6 RNA polymerase promoter sequence.

The percentage of base similarity is the total number of bases in acontiguous base sequence which are the same as those present in a fulllength RNA polymerase promoter sequence, divided by the number of basesin the full length RNA polymerase sequence. Thus, with respect to the T7polymerase provided in SEQ. ID. NO: 3, the number of bases present inthe decoy probes that are the same as T7 polymerase promoter sequence isdivided by 23. For example, SEQ. ID. NO: 7: taatacgact cactataggg, has asequence similarity of about 87% to the T7 polymerase provided in SEQ.ID. NO: 3. Changes to the bases listed for SEQ. ID. NO: 7 would affectthe sequence similarity.

Another way to design decoy probes is to use a sequence having a regionof self-complementarity which will produce a stem loop structure. Inaddition to recognizing promoter sequences, RNA polymerases also appearto recognize secondary structures.

In addition to having a sequence targeted to an RNA polymerase, decoyprobes can include additional sequences. Additional sequences can berandomly generated. Preferably, additional sequences are notcomplementary to target nucleic acid or the complement thereof, or anyother sequence present in the amplification reaction. In an embodimentof the present invention, a decoy probe contains an additional sequenceto provide the decoy probe with a total length approximating the totallength of an amplification oligonucleotide used in an amplificationprotocol (e.g., ±five bases).

B. The Protein Binding Amplification Protocol

The Protein Binding Amplification Protocol can be used to select for,and to produce large numbers of, an oligonucleotide able to bind to aprotein. Preferably, the protocol is performed using an amplificationenzyme such as an RNA polymerase or a reverse transcriptase. Severaltechniques useful in carrying out the Protein Binding AmplificationProtocol are described by Gold et al., Amu. Rev. Biochem., 64:763-797(1995), and Uphoff et al., Curr. Opin. Struct. Biol., 6:281-288 (1996),both of which are hereby incorporated by reference herein.

The Protein Binding Amplification Protocol can be performed using acollection of two or more oligonucleotides, each comprising a known 3′region, a potential protein binding region, and a known 5′ region. Thepotential protein binding region can be a randomly generated sequenceregion.

The collection of oligonucleotides is combined with the protein to allowfor protein binding. Protein binding oligonucleotides are then separatedfrom unbound oligonucleotides. Separation can be achieved usingdifferent techniques, such as immunoprecipitation using an antibody thatrecognizes the protein.

Another example of a separation technique uses a protein bound to anaffinity column, where oligonucleotides not bound to the protein can bewashed through the column under conditions where protein boundoligonucleotides are retained. The bound oligonucleotides can then beseparated from the protein.

The oligonucleotides that bind to the protein are combined with apromoter-template complementary probe under conditions where adouble-stranded promoter region is produced. The promoter-templatecomplementary probe hybridizes to a portion of the oligonucleotides'known 3′ end, followed by the formation of a double-stranded promoter.

A double-stranded promoter can be formed by different techniques, suchas by hybridization with a complementary promoter sequence or byproducing a complementary promoter sequence using DNA polymeraseactivity. Preferably, a double-stranded promoter is created using a DNApolymerase by extending the 3′ end of an oligonucleotide using thepromoter sequence of the promoter-template complementary probe as atemplate.

The oligonucleotide joined to the double-stranded promoter is used in atranscription-associated amplification reaction to produce multiple RNAtranscripts. The produced transcripts are complementary to the proteinbinding oligonucleotide.

The RNA transcripts are used as a template to produce multiple copies ofthe protein binding oligonucleotide in primer extension reactions.Primer extension reactions are performed using a primer analogous to theknown 5′ end of the oligonucleotide (and thus complementary to the 3′end of the RNA transcript) and a DNA polymerase under DNA polymeraseamplification conditions. Preferably, the DNA polymerase is a reversetranscriptase. RNA which is present in an RNA:DNA duplex can be removedusing RNase H activity. Multiple cycles can be carried out to producelarger numbers of oligonucleotides binding to the protein and to selectfor higher affinity polymerase binding sequences.

C. Decoy Probe Construction

Decoy probes are nucleotide base sequence recognition moleculescomprising nucleotide base recognition groups joined together by abackbone. The different subunits of the decoy probe should allow thedecoy probe to competitively and reversibly bind to an amplificationenzyme. Components which should be avoided include those which preventbinding to an amplification enzyme and which result in irreversibleinhibition of enzymatic activities.

A given nucleotide base recognition group present in a nucleotide basesequence recognition molecule may be complementary to a particularnucleotide (e.g., adenine, guanine, cytosine, thymine, and uracil), andthus, be able to hydrogen bond with that nucleotide. A nucleotide baserecognition group may also be able to hydrogen bond with differentnucleotides. For example, when inosine is a nucleotide base recognitiongroup it can hydrogen bond with different nucleotide bases.

Preferred nucleotide base recognition groups are nitrogenous purine orpyrimidine bases, or derivatives thereof, able to hydrogen bond witheither adenine, guanine, cytosine, thymine or uracil. Examples of suchrecognition groups include adenine, guanine, cytosine, thymine, uracil,and derivatives thereof. Examples of derivatives include modified purineor pyrimidine bases such as N⁴-methyl deoxyguanosine, deaza or azapurines and pyrimidines used in place of natural purine and pyrimidinebases, pyrimidine bases having substituent groups at the 5 or 6position, and purine bases having an altered or a replacementsubstituent at the 2, 6 or 8 positions. See, e.g., Cook, InternationalApplication No. PCT/US92/11339, International Publication No. WO93/13121 (hereby incorporated by reference herein). Additional examplesinclude, 2-amino-6-methylaminopurine, O6-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, O4-alkyl-pyrimidines (see, e.g., TheGlen Report, Volume 1 (1993), hereby incorporated by reference herein).

A nucleotide base sequence recognition molecule backbone can be made upof different groups. Examples of different groups includesugar-phosphodiester type backbone groups and peptide nucleic acidbackbone groups.

Structure I illustrates a sugar-phosphodiester type backbone where thesugar group is a pentofuranosyl group. The sugar groups are joinedtogether by a linkage such as a phosphodiester linkage or other suitablelinkage.

X represents the group joining two sugars. Examples of X include—OP(O)₂O—, —NHP(O)₂O—, —OC(O)₂O—, —OCH₂C(O)₂NH—, —OCH₂C(O)₂O—,—OP(CH₃)(O)O—, —OP(S)(O)O— and —OC(O)₂NH—. As with the other examplesprovided herein, other equivalents that are well known in the art orwhich become available can also be used.

Y₁ and Y₂ are independently selected groups. Examples of Y₁ and Y₂include H, OH, C₁-C₄ alkoxy, halogen, and C₁-C₆ alkyl. Preferably, Y₁and Y₂ are independently either H, OH, F, or OCH₃. C₁-C₆ alkyl and C₁-C₄alkoxy, may be or may include groups which are straight-chain, branched,or cyclic.

Base₁ and Base₂ are independently selected from the group consisting of:adenine, guanine, cytosine, thymine, uracil, or a group that does notinhibit complementary base pairing of an adjacent base to acomplementary nucleic acid. Examples, of groups not inhibitingcomplementary base pairing include smaller size groups such as hydrogen,OH, C₁-C₆ alkyl, and C₁-C₄ alkoxy. In different embodiments, thenucleotide base recognition sequence contains at least about 7, or atleast about 10, and no more than about 40, or about 30, basesindependently selected from the group consisting of: adenine, guanine,cytosine, thymine, and uracil.

R₁ and R₂ represent independently selected groups. Examples of R₁ and R₂include additional sugar-phosphodiester type groups, hydrogen, hydroxy,peptide nucleic acid, phosphate, thiophosphate, C₁-C₆ alkyl, andmolecules not providing sequence information such as polysaccharides,polypeptides, peptides, and other non-nucleotide linkages.

Derivatives of Structure I able to be a component of a nucleotide baserecognition sequence are well known in the art and include, for example,molecules having a different type of sugar. For example, a nucleotidebase recognition sequence can have cyclobutyl moieties connected bylinking moieties, where the cyclobutyl moieties have hetereocyclic basesattached thereto. See, e.g., Cook et al., International Application No.PCT/US93/01579, International Publication No. WO 94/19023 (herebyincorporated by reference herein).

In an embodiment of the present invention, a nucleotide base recognitionmolecule is a polynucleotide or derivative thereof. A “polynucleotide orderivative thereof” is a nucleotide base recognition molecule made up ofstructure I repeating units where X is —OP(O)₂O—; Y₁ and Y₂ areindependently selected groups from the group consisting of H, OH, OCH₃,and F; Base₁ and Base₂ are independently selected from the groupconsisting of: adenine, guanine, cytosine, thymine, and uracil. Theterminal portion of the molecule contains R₁ and R₂ independentlyselected from the group consisting of OH, C₁-C₆ alkyl, phosphate,thiophosphate.

Another type of a nucleotide base sequence recognition molecule backboneis that present in peptide nucleic acid. Peptide nucleic acid in a DNAanalogue where the deoxyribose phosphate backbone is replaced by apseudo peptide backbone. Peptide nucleic acid is described by Hyrup andNielsen, Bioorganic & Medicinal Chemistry, 4:5-23 (1996), andHydig-Hielsen and Godskesen, International Application No.PCT/DK95/00195, International Publication No. WO 95/32305, each of whichis hereby incorporated by reference herein.

Preferably, the peptide nucleic acid is made up ofN-(2-aminoethyl)glycine units as illustrated in Structure II.

R₁, R₂, and Base₁ is as described for Structure I type compounds.

Nucleotide base sequence recognition molecules can be produced usingstandard techniques. Publications describing organic synthesis ofoligonucleotides and modified oligonucleotides include Eckstein, F.,Oligonucleotides and Analogues, A Practical Approach, Chapters 1-5(1991), which reviews organic synthesis of oligonucleotides; Carutherset al., In Methods In Enzymology 154:287 (1987), which describes aprocedure for organic synthesis of oligonucleotides using standardphosphoramidite solid-phase chemistry; Bhatt, U.S. Pat. No. 5,252,723,which describes a procedure for organic synthesis of modifiedoligonucleotides containing phosphorothioate linkages; and Klem et al.,International Publication NO. WO 92/07864, which describes organicsynthesis of modified oligonucleotides having different internucleosidelinkages including methylphosphonate linkages. (Each of these referencesis hereby incorporated by reference herein.)

Additional references describing techniques which can be used to producedifferent types of nucleotide base sequence recognition moleculesinclude Cook, International Application No. PCT/US92/11339,International Publication No. WO 93/13121; Miller et al., InternationalApplication No. PCT/US94/00157, International Publication No. WO94/15619; McGee et al., International Application No. PCT/US93/06807,International Publication No. WO 94/02051; Cook et al., InternationalApplication No. PCT/US93/01579, International Publication No. WO94/19023; Hyrup and Nielsen, Bioorganic & Medicinal Chemistry, 4:5-23(1996); and Hydig-Hielsen and Godskesen, International Application No.PCT/DK95/00195, International Publication No. WO 95/32305. (Each ofthese references is hereby incorporated by reference herein.)

Decoy probes preferably contain two regions: (1) a first region which ispreferably a polymerase binding region or a promoter similar region; and(2) a second region which is not substantially complementary to nucleicacid used in an amplification protocol.

In an embodiment of the present invention, the first region comprises(a) a backbone containing one or more groups independently selected fromthe group consisting of one or more sugar-phosphodiester type groups andone or more peptide nucleic acid groups, and (b) at least ten nucleotidebase recognition groups joined to the backbone, wherein each recognitiongroup can independently hydrogen bond with at least one of adenine,guanine, cytosine, thymine or uracil. In additional embodiments, atleast about 15, at least about 20, or at least about 25 recognitiongroups are present.

In another embodiment of the present invention, the second regioncomprises (a) a backbone containing one or more groups independentlyselected from the group consisting of one or more sugar-phosphodiestertype groups and one or more peptide nucleic acid groups, and (b) atleast five nucleotide base recognition groups joined to the backbone,wherein each recognition group can independently hydrogen bond with atleast one of adenine, guanine, cytosine, thymine or uracil. Inadditional embodiments, at least about 10, at least about 15, or atleast about 20 recognition groups are present.

In a preferred embodiment, the decoy probe is made up of optionallymodified oligonucleotides. Optionally modified oligonucleotides maycontain altered sugar groups, altered phosphodiester linkages, and/oraltered nitrogenous bases. Preferred modifications include differentpurine or pyrimidine nitrogenous bases, or derivatives thereof, able tohydrogen bond to either adenine, guanine, thymine or cytosine; differentsugar moieties such as 2′ alkoxy ribose, 2′ halo ribose and cyclobutyl;and different internucleoside linkages such as methylphosphonate, andphosphorothioate. Preferably, the 2′ alkoxy ribose, if present, is 2′methoxy ribose, and the 2′ halo ribose, if present, is 2′ flouro ribose.

In a preferred embodiment, the decoy probe containing a first and asecond region consists of 15 to 100 optionally modified nucleosides andone or more blocking groups located at the 3′ terminus of the probe.Preferably, each of the optionally modified nucleosides independentlyhas a purine or pyrimidine moiety independently selected from the groupconsisting of inosine, uracil, adenine, guanine, thymine and cytosine;and a sugar moiety independently selected from the group consisting ofdeoxyribose, 2′-methoxy ribose, and ribose; and each of the optionallymodified nucleosides is joined together by an internucleoside linkageindependently selected from the group consisting of phosphodiester,phosphorothioate, and methylphosphonate. More preferably, the firstregion is covalently joined at its 5′ end to the 3′ end of the secondregion through a phosphodiester, phosphorothioate, methylphosphonate, orpolysaccharide group. More preferably, the probe contains 15 to 75, evenmore preferably 35-70 optionally modified nucleosides.

In a more preferred embodiment, at least 80% of the optionally modifiednucleosides have a purine or pyrimidine moiety independently selectedfrom the group consisting of adenine, guanine, thymine and cytosine; anda deoxyribose sugar moiety; and at least 80% of internucleoside linkagesjoining the optionally modified nucleosides are phosphodiester. Evenmore preferably, the probe consists of independently selecteddeoxyribonucleotides and one or more blocking groups.

D. Decoy Probe Configurations

Decoy probes can be designed with a first and a second nucleotide baserecognition sequence region having different configurations. Examples ofdifferent configurations include the 3′ or 5′ end of a first nucleicacid region being joined directly, or through a linker, to either the 5′or 3′ end of a second nucleic acid region.

FIGS. 2A and 2B provide illustrations of two different decoy probeconfigurations. The first nucleotide base recognition sequence region isunderlined in FIGS. 2A and 2B. FIG. 2A illustrates a decoy probe where a5′ end of the first region is joined directly to a 3′ end of the secondregion. FIG. 2B illustrates a decoy probe where the 5′ end of the firstregion is joined to the 5′ end of the second region through anon-nucleotide linker (indicated by the curved line). The terminalpropyl groups shown in FIGS. 2A and 2B are blocking groups.

E. Blocking Groups

Blocking groups are chemical moieties which can be added to a nucleicacid to inhibit nucleic acid polymerization catalyzed by a nucleic acidpolymerase. Blocking groups are typically located at the terminal 3′end(s) of a decoy probe which is made up of nucleotides or derivativesthereof. By attaching a blocking group to a terminal 3′ OH, the 3′ OHgroup is no longer available to accept a nucleoside triphosphate in apolymerization reaction.

Numerous different groups can be added to block the 3′ end of a probesequence. Examples of such groups include alkyl groups, non-nucleotidelinkers, phosphorothioate, alkane-diol residues, peptide nucleic acid,and nucleotide derivatives lacking a 3′ OH (e.g., cordycepin).

An alkyl blocking group is a saturated hydrocarbon up to 12 carbons inlength which can be a straight chain or branched, and/or contain acyclic group. More preferably, the alkyl blocking group is a C₂-C₆ alkylwhich can be a straight chain or branched, and/or contain a cyclicgroup.

IV. Reagent Mixtures and Kits

The present invention also features a reagent mixture containing anamplification enzyme and a reversible inhibitor of the enzyme.Preferably, the reagent mixture does not contain amplificationoligonucleotides and/or target nucleic acid.

Reagent mixtures can be packaged as part of a kit providing anamplification enzyme for use in an amplification reaction. Such kits mayalso contain other components of an amplification reaction, generally,in the same or different compartments. Additional components can includedeoxyribonucleoside triphosphates, preferably deoxyriboadenosinetriphosphate, deoxyribothymidine triphosphate, deoxyriboguanosinetriphosphate, and deoxyribocytosine triphosphate; ribonucleosidetriphosphates, preferably, riboadenosine triphosphate, ribouridinetriphosphate, riboguanosine triphosphate, and ribocytosine triphosphate;buffers suitable for amplification reactions; and enzymes used in theamplification reaction.

Amplification oligonucleotides, and/or labeled nucleic acid probes whichcan be used to detect amplification products may also be included as kitcomponents. Such components are preferably in separate compartment(s)from amplification enzymes.

In a preferred embodiment, the reagent mixture is suitable for providingcomponents to be used for a transcription-associated amplification andincludes an RNA polymerase and/or a reverse transcriptase. Morepreferably, the reagent mixture does not contain unblockedoligonucleotides and/or does not contain an oligonucleotide comprising a5′ promoter sequence.

V. Amplification Procedures

The present invention can be used in conjunction with differentamplification procedures. Applicable procedures are those involving theuse of a nucleic acid polymerase. By combining a nucleic acid polymerasewith a reversible inhibitor, such as a decoy probe, the ability of thepolymerase to form undesirable products may be inhibited.

The “BACKGROUND OF THE INVENTION” section supra, provides examples ofamplification procedures involving the use of DNA and/or RNApolymerases. Suitable DNA and/or RNA polymerases for carrying out theseand other amplification procedures involving nucleic acid polymerizationare readily available, and include for example, DNA-dependent DNApolymerases such as DNA polymerase I, T4 DNA polymerase, Taq polymeraseand exonuclease deficient klenow; DNA-dependent RNA polymerase such asT7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase; andRNA-dependent DNA polymerases such as avian myeloblastosis virus (AMV)reverse transcriptase, Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, and HIV reverse transcriptase.

An advantage of the present invention is that it provides a means forreducing undesirable side-products under isothermal conditions. Thisadvantage is particularly suited for different protocols, such astranscription-associated amplification and SDA which can be carried outusing isothermal conditions. The “BACKGROUND OF THE INVENTION” sectionsupra, provides different formats that can be employed for carrying outtranscription-associated amplification and SDA. Examples of thedifferent formats include, Burg et al., U.S. Pat. No. 5,437,990, whichincludes a description of a general transcription-associatedamplification format; and Kacian et al., U.S. Pat. No. 5,399,491, whichincludes a description of a transcription-associated amplificationformat featuring the use of RNase H activity present in reversetranscriptase to achieve strand separation. (Each of these references ishereby incorporated by reference herein.)

Additional procedures referenced in “BACKGROUND OF THE INVENTION”section supra, include Kacian et al., U.S. Pat. No. 5,554,516; Kacian etal., International Application No. PCT/US93/04015, InternationalPublication No. WO 93/22461; Gingeras et al., International ApplicationNo. PCT/US87/01966, International Publication No. WO 88/01302; Gingeraset al., International Application No. PCT/US88/02108, InternationalPublication No. WO 88/10315; Davey and Malek, European Application No.88113948.9, European Publication No. 0 329 822 A2; Malek et al., U.S.Pat. No. 5,130,238; Urdea, International Application No. PCT/US91/00213,International Publication No. WO 91/10746; McDonough et al.,International Application No. PCT/US93/07138, International PublicationNo. WO 94/03472; Ryder et al., International Application No.PCT/US94/08307, International Publication No. WO 95/03430; Walker, PCRMethods and Applications, 3:25-30 (1993); Walker et al., Nucleic AcidsRes., 20:1691-1696 (1992); and Walker et al. Proc., Natl. Acad. Sci.,89:392-396 (1991). (Each of these references is hereby incorporated byreference herein.)

Preferably, amplification of a nucleic acid sequence is carried out byfirst combining together an amplification enzyme with a reversibleinhibitor of the enzyme in the absence of amplification oligonucleotidesable hybridize to target nucleic acid. After the combining step, theenzyme combined with the inhibitor is then used in an amplificationreaction.

The first combining step may be thought of as a pre-incubation stepallowing the reversible inhibitor to bind to, or otherwise sequester, anamplification enzyme. Preferably, the reversible inhibitor is a decoyprobe. More preferably, the amplification procedure is eithertranscription-associated amplification or SDA.

In a preferred embodiment concerning transcription-associatedamplification, the amplification enzyme(s) are an RNA polymerase and/orreverse transcriptase, and the pre-incubation occurs in the absence ofpromoter-target complementary oligonucleotides. More preferably, thepre-incubation occurs in the absence of both primers and promoter-targetcomplementary oligonucleotides.

In preferred embodiments directed towards SDA, the amplification enzymeis a DNA polymerase lacking 5′-3′ exonuclease activity, and thepre-incubation occurs in the absence of SDA amplificationoligonucleotides.

EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the present invention. These examples are not intended tolimit the claimed invention.

I. HIV Amplification Conditions

With the exception of varying target concentration, standard T7 HIVamplification reactions contained 15 pMol/reaction of a T7promoter-primer, 15 pMol/reaction of an analogous primer, 35 mM KCl, 75mM Tris-Cl pH 7.5, 9 mM HEPES pH 7.5, 20 mM MgCl₂, 1 mM dATP, 1 mM dCTP,1 mM dGTP, 1 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 5% w/vPVP, 0.15 mM ZnOAc, 10% v/v glycerol, 12.5 mM NALC, 0.75 mM EDTA, 2.5%Triton® X-102 (Sigma, St. Louis, Mo.), 0.0025% phenol red, 100-200Epicentre units of reverse transcriptase (Epicentre Technologies Inc.,Madison, Wis.) and about 500 Epicentre units of T7 RNA polymerase(Epicentre Technologies Inc.) in either 20 μL or 100 μL reaction volume,unless otherwise noted.

Standard T3 HIV amplification reactions contained 15 pMol/reaction of aT3 promoter-primer with 15 pMol/reaction of an analogous primer, 35 mMKCl, 2.5 mM NaCl, 65 mM Tris-Cl pH 7.5, 20 mM MgCl₂, 1 mM dATP, 1 mMdCTP, 1 mM dGTP, 1 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 2.5%v/v glycerol, 10 mM DTT, 0.25 mM EDTA, 0.0025% Triton® X-100 (Sigma),100-200 Epicentre units of reverse transcriptase, transcriptase(Epicentre Technologies Inc.), and 500 Epicentre units T3 RNA polymerase(Epicentre Technologies Inc.) in either 20 μl or 100 μl reaction volume,unless otherwise noted.

For 100 μl amplification reactions (T7 or T3 amplification reactions),25 μl of amplification reagent was aliquoted to individual tubes,followed by the addition of 200 μl of mineral oil. Target RNA(synthesized by in vitro transcription reactions) was diluted to theappropriate copy number in water and added in a 50 μl volume. Reactionswere incubated at 60° C. (in a dry bath incubator or waterbath) for 10minutes, followed by an incubation at 42° C. for 5 minutes. Twenty-fivemicroliters of enzyme reagent, containing reverse transcriptase and T7or T3 RNA polymerase with or without decoy probes, was then added andthe reaction tubes were incubated at 42° C. for an additional 60-90minutes, except for the experiments examining transcription-associatedamplification kinetics in which the reactions were terminatedprematurely. Reactions were terminated by the addition of HPA probereagent which was the initial step in the amplicon detection method. For20 μl transcription-associated amplification reactions (17 or T3), allreagents were added at one fifth volume described for 100 μl reactions.

II. HPA Detection

Amplicon production was detected by hybridization with acridinium esterlabeled oligonucleotide detection probes. (See, e.g., Arnold et al.,U.S. Pat. No. 5,283,174 hereby incorporated by reference herein.) Insome instances, one or more unlabeled helper oligonucleotides were usedto facilitate hybridization to the nucleic acid having the targetsequence. (See, e.g., Hogan et al., U.S. Pat. No. 5,030,557, herebyincorporated by reference herein.)

Hybridization of the detection probes was performed in HPA probe reagentmade up of 0.05 M lithium succinate pH 5, 0.6 M lithium chloride,1%(w/v) lithium lauryl sulfate (LLS), 10 mM EDTA, 7.5 mM aldrithiol and10 mM EGTA at 60° C. for 10 minutes. HPA probe reagent was normally madeas a 2×stock containing detection probe and an equal volume was added toeach amplification reaction. Following a 10 minute hybridization at 60°C., 300 μl (3×reaction volume) of a solution containing 0.15 M sodiumtetraborate pH 8.5, and 1% Triton® X-100 was added to each tube and thereactions were incubated at 60° C. for an additional 15 minutes.

Detection and quantitation of hybrid molecules were accomplished using aluminometer (e.g., LEADER™ 50; Gen-Probe Incorporated, San Diego,Calif.). The luminometer automatically injects two reagents, the firstbeing composed of 1 mM nitric acid and 1% hydrogen peroxide, the secondbeing composed of 1 N sodium hydroxide. The reagents cause the formationof chemiluminescence from unaltered acridinium esters present inacridinium ester labeled oligonucleotides. Assay results were given inRelative Light Units (RLUs), a measure of the number of photons detectedby the luminometer.

III. Nucleic Acid Sequences

The following nucleic acid sequences are examples of decoy probes thatmay be used in the present invention:

SEQ. ID. NO: 8: gtactcagat gctgcactga aattattaac cctcactaaa gggatataa;SEQ. ID. NO: 9: gtactcagat gctgtcactg atcataatac gactcactat agggagataa;SEQ. ID. NO: 10: gtactcagat gctgcactga aatcaattcg actcactaaa gggatataa;SEQ. ID. NO: 11: gtactcagat gctgcactga aatcaattcg actcactaaa tccatataa;SEQ. ID. NO: 12: gtactcagat gctgcactga aattaatacg actcactata gccatataa;SEQ. ID. NO: 13: gaaatcaatt cgactcacta aagggatata a; and SEQ. ID. NO:14: gtactcagat gctgtcactg atcagtactc agatgctgtg atgcactgat caaa.

The bold portion of SEQ. ID. NOs. 8-13 refers to promoter-similarsequence regions. SEQ. ID. NO: 14 is a random sequence. Decoy probesused in the examples described below were blocked at the terminal 3′ OHby a n-propyl group.

Example 1 Use of Decoy Probes in a T7 Amplification

Decoy probes containing sequences similar, or identical, to an RNApolymerase native promoter were tested for their ability to enhanceamplification using the T7 RNA polymerase transcription-associatedamplification system. A promoter-primer and a complementary primer wereused in a standard transcription-associated amplification reaction (20μL volume) to amplify 20 copies of HIV target RNA in the presence orabsence of 10-20 pMol of 3′ blocked oligonucleotides of SEQ. ID. Nos. 8,9, 10, or 14.

Amplicon produced from the reactions was quantitated by HPA with anacridinium ester labeled detection probe at a concentration of 0.1 pMolper reaction. The number of reactions producing a positive amplificationwere scored. An amplification was positive if ≧30,000 RLU was observedduring the HPA detection step. The 30,000 RLU value is over 30-foldabove background signal and represents a minimum of 10⁹-fold targetamplification. The results are shown in Table 1.

TABLE 1 Summary of results from experiments examining the effects ofdifferent decoy probes on T7 transcription-associated amplificationperformance using 20 copies of target (20 μL reaction volume). Reactionswere considered positive when RLUs were ≧30,000. T7Transcription-Associated Amplification NO SEQ. ID. SEQ. ID. SEQ. ID.SEQ. ID. DECOYS NO: 8 NO: 10 NO: 9 NO: 14 Amount of (10-20 (10-20 (10-20(6-20 Oligonu- pMol/rxn) pMol/rxn) pMol/rxn) pMol/rxn) cleotide Total144 128 248 208 72 Reactions Number of  94  74 222 164 33 PositivesPercent 67% 58% 90% 79%  46% Sensitivity

A higher positivity rate is equivalent to a higher sensitivity. The bestresults were obtained with the SEQ. ID. NO: 10 decoy probe containing asequence similar, but not identical, to a T3 and T7 RNA polymerasepromoter. The SEQ. ID. NO: 10 decoy probe has 16/23 matches to the T3consensus sequence and 19/23 matches to the T7 consensus sequence. Thepositivity rate increased from 67% without decoy probes to 90% with theSEQ. ID. NO: 10 decoy probe. Reactions that included a decoy probecontaining a consensus T7 promoter sequence (SEQ. ID. NO: 9) producedhigher sensitivity than reactions with no decoy probe, or decoy probeswithout a promoter-similar sequence, but not as good as reactions withthe SEQ. ID. NO: 10 decoy probe.

Example 2 Use of Decoy Probes in a T3 Amplification

Decoy probes containing sequences similar, or identical, to an RNApolymerase native promoter were tested for their ability to enhanceamplification using the T3 RNA polymerase transcription-associatedamplification system. T3 transcription-associated amplificationreactions were similar to those described for Example 1, with theprimary exception being the use of a T3 promoter-primer and T3 RNApolymerase. Twenty copies of HIV target RNA were initially present (20μL reaction volume). The results are shown in Table 2.

TABLE 2 Summary of results from experiments examining the effects ofdecoy probes on T3 transcription-associated amplification performance.Identity and quantity of each decoy probe added to the T3 enzyme reagentin 20 μL reaction volume are shown. Reactions were considered positivewhen RLUs were ≧30,000. T3 Transcription-Associated Amplification NOSEQ. ID. SEQ. ID. SEQ. ID. SEQ. ID. DECOYS NO: 8 NO: 10 NO: 9 NO:14Amount of (10-20 (10-20 (10-20 (6-20 Oligo- pMol/rxn) Pmol/rxn)pMol/rxn) pMol/rxn) nucleotide Total 256 160 224 144 40 Reactions Numberof 126 104 166 104 20 Positives Percent  49%  65%  74%  73%  50%Sensitivity

As with Example 1, the best results were obtained with the SEQ. ID. NO:10 decoy probe containing a sequence similar, but not identical, to a T3or T7 RNA polymerase promoter. Reactions that included a decoy probecontaining a T3 promoter sequence (SEQ. ID. NO: 8), or the T7 promotersequence (SEQ. ID. NO: 9) produced higher sensitivity than reactionswith no decoy probe, or decoy probes without a promoter-similarsequence.

Example 3 Decoy Probe Length

Decoy probe length was examined using the SEQ. ID. NO: 13 probe that isa truncated version of the SEQ. ID. NO: 10 probe. The ability of theSEQ. ID. NO: 13 probe to enhance amplification was measured using T7amplification and detection as described in Example 1. Table 3,summarizes the results. A comparison of the results in Table 2 and Table3 indicates that the shorter length SEQ. ID. NO: 13 probe significantlyenhanced transcription-associated amplification performance, though to alesser extent than the SEQ. ID. NO: 10 probe.

TABLE 3 Summary of results from experiments examining the effects of atruncated decoy probe on T7 transcription-associated amplification.Twenty copies of HIV RNA target were initially present in 20 μL reactionvolume. Reactions were considered positive when RLUs were ≧30,000. T7Transcription-Associated Amplification No Decoy SEQ. ID. NO: 13 Amountof Oligonucleotide (20-22 pMol/rxn) Total Reactions 64 64 Number ofPositives 37 44 Percent Sensitivity  59%  69%

Example 4 Use of Decoy Probes on Additional Targets

This example confirms that the amplification enhancement observed usingdecoy probes is not limited to a particular type of target nucleic acid.T7 transcription-associated amplification reactions were performed using20 copies of a HCV target under conditions similar to those describedfor T7 HIV transcription-associated amplification reactions inExample 1. The primary exception to the Example 1 amplificationconditions was the use of HCV sequence-specific amplificationoligonucleotides.

Reactions contained 18 pMol of a T7 promoter-primer along with 10pMol/reaction of analogous primers, and 20 copies of target. Ampliconproduced from the reactions was quantitated by HPA as described inExample 1, except that acridinium ester-labeled probes at aconcentration of 0.05 pMol each per reaction, with helper probes at aconcentration of 2.5 pMol each per reaction, were used.

The addition of decoy probe SEQ. ID. NO: 10 to the enzyme reagentsignificantly improved the performance of this assay. The percentage ofpositive reactions containing 10 copies of HCV RNA increased from 78%(25/32) to 100% (25/25) in the presence of decoy probes of SEQ. ID. NO:10.

Example 5 Decoy Probe Kinetics Using HCV and T7

Decoy probes increase the kinetics of T7 HCV transcription-associatedamplification reactions. T7 HCV reactions and detection were performedas described in Example 4, except that the amount of amplicon producedwas quantified as a function of amplification time. Amplicon producedfrom the reactions was quantitated by HPA with HCV probes as describedfor Example 4, and a positive was scored when a signal of 30,000 RLU orgreater was obtained. The results are shown in FIG. 3.

The addition of 22 pMol/rxn of decoy probe SEQ. ID. NO: 10 to 20 μL HCVtranscription-associated amplification reactions containing 20 copies oftarget RNA, increased the rate of amplicon production. Ten minutes afterthe addition of enzyme reagent, 75% (30/40) of the reactions containingdecoy probes were positive (≧30,000 RLUs) whereas only 10% (4/40) of thereactions lacking decoy probes were positive. Furthermore, after a 10minute amplification time, the average RLUs for the samples containingdecoy probe was 4-fold higher (64,951 RLUs) than those lacking it(16,317 RLUs).

Example 6 Decoy Probe Kinetics Using HIV and T7

This example illustrates the ability of decoy probes to increase thekinetics of the T7 HIV transcription-associated amplification system. T7HIV transcription-associated amplification reactions were performed inthe presence or absence of decoy probe SEQ. ID. NO: 10. Amplificationand amplicon detection were carried out as described in Example 1. Theresults, illustrated in FIG. 4, show that the average signal increasedat a faster rate as did the average RLU values for the samplepopulation.

Example 7 Decoy Probe Kinetics Using HIV and T3

This example illustrates the ability of decoy probes to increase thekinetics of the T3 HIV transcription-associated amplification system. T3HIV transcription-associated amplification reactions were performed inthe presence or absence of decoy probe SEQ. ID. NO: 10. Amplificationand amplicon detection were carried out as described in Example 2.

The results, illustrated in FIG. 5, show that the percentage of positivereactions increased at a faster rate as did the average RLU values forthe sample population when a decoy probe was used.

Other embodiments are within the following claims. Thus, while severalembodiments have been shown and described, various modifications may bemade, without departing from the spirit and scope of the presentinvention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 14 <210> SEQ ID NO 1 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: synthetic construct <400> SEQUENCE: 1taatattaac cctcactaaa gggaga           #                  #              26 <210> SEQ ID NO 2 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 2tctcccttta gtgagggtta atatta           #                  #              26 <210> SEQ ID NO 3 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 3taatacgact cactataggg aga            #                  #                23 <210> SEQ ID NO 4 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 4tctccctata gtgagtcgta tta            #                  #                23 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 5atttaggtga cactatagaa gag            #                  #                23 <210> SEQ ID NO 6 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 6ctcttctata gtgtcaccta aat            #                  #                23 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 7taatacgact cactataggg             #                  #                   # 20 <210> SEQ ID NO 8 <211> LENGTH: 49<212> TYPE: DNA <213> ORGANISM: synthetic construct <400> SEQUENCE: 8gtactcagat gctgcactga aattattaac cctcactaaa gggatataa  #               49 <210> SEQ ID NO 9 <211> LENGTH: 50 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 9gtactcagat gctgtcactg atcataatac gactcactat agggagataa  #              50 <210> SEQ ID NO 10 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 10gtactcagat gctgcactga aatcaattcg actcactaaa gggatataa  #               49 <210> SEQ ID NO 11 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 11gtactcagat gctgcactga aatcaattcg actcactaaa tccatataa  #               49 <210> SEQ ID NO 12 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 12gtactcagat gctgcactga aattaatacg actcactata gccatataa  #               49 <210> SEQ ID NO 13 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 13gaaatcaatt cgactcacta aagggatata a         #                  #          31 <210> SEQ ID NO 14 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: synthetic construct <400> SEQUENCE: 14gtactcagat gctgtcactg atcagtactc agatgctgtg atgcactgat ca#aa           54

What is claimed is:
 1. A purified decoy probe comprising: a firstnucleotide base recognition sequence region, wherein said first regionbinds to an RNA polymerase; and an optionally present second nucleotidebase recognition sequence region, provided that if said first region isnucleic acid and said second region is present, then said second regionis either directly joined to the 5′ end of said first region is joinedto the 3′ end or 5′ end of said first region by a non-nucleotide linker,wherein said optionally present second region is present if said firstregion can be used to produce a functional double-stranded promotersequence using a complementary oligonucleotide, further provided that ifsaid first region is nucleic acid which can be used to produce saidfunctional double-stranded promoter sequence using said complementaryoligonucleotide, then said decoy probe does not have a nucleic acidsequence greater than about 10 nucleotides in length joined directly tothe 3′ end of said first region and said decoy probe does not have aterminal 3′ OH group available to accept a nucleoside triphosphate in apolymerization reaction.
 2. The probe of claim 1, wherein said firstregion is nucleic acid.
 3. The probe of claims 1, wherein said probeconsists of 15 to 100 optionally modified nucleosides and one or moreblocking groups located at the 3′ terminus of said probe, wherein eachof said optionally modified nucleosides independently has, a purine orpyrimidine moiety independently selected from the group consisting ofinosine, uracil, adenine, guanine, thymine and cytosine; and a sugarmoiety independently selected from the group consisting of deoxyribose,2′-methoxy ribose, and ribose; and each of said optionally modifiednucleosides is joined together by an internucleoside linkageindependently selected from the group consisting of phosphodiester,phosphorothioate, and methylphosphonate.
 4. The probe of claim 3,wherein at least 80% of said optionally modified nucleosides have apurine or pyrimidine moiety independently selected from the groupconsisting of adenine, guanine, thymine and cytosine, and a deoxyribosesugar moiety; and at least 80% of said internucleoside linkages joiningsaid optionally modified nucleosides are phosphodiester.
 5. The probe ofclaim 4, wherein said probe consists of 15 to 100 independently selecteddeoxyribonucleotides and one or more blocking groups located at the 3′terminus of said probe.
 6. The probe of claim 3, wherein said one ormore blocking groups are selected from the group consisting ofphosphorothioate, alkane-diol residue, cordycepin, and an alkyl group.7. The probe of claim 6, wherein said probe consists of 35 to 70independently selected nucleotides and said one or more blocking groups.8. The probe of claim 7, wherein said RNA polymerase is T7 RNApolymerase.
 9. The probe of claim 7, wherein said RNA polymerase is T3RNA polymerase.
 10. The probe of claim 7, wherein said RNA polymerase isSP6 RNA polymerase.
 11. A purified decoy probe comprising: a firstnucleotide base recognition sequence region, wherein said first regionhas at least 35% sequence similarity to an RNA polymerase promotersequence; and an optionally present second nucleotide base recognitionsequence region, provided that if said first region is nucleic acid andsaid second region is present, then said second region is eitherdirectly joined to the 5′ end of said first region or is joined to the3′ end or 5′ end of said first region by a non-nucleotide linker,wherein said optionally present second region is present if said firstregion can be used to produce a functional double-stranded promotersequence using a complementary oligonucleotide, further provided that ifsaid first region is nucleic acid which can be used to produce saidfunctional double-stranded promoter sequence using said complementaryoligonucleotide, then said decoy probe does not have a nucleic acidsequence greater than about 10 nucleotides in length joined directly tothe 3′ end of said first region and said decoy probe does not have aterminal 3′ OH group available to accept a nucleoside triphosphate in apolymerization reaction.
 12. The probe of claim 11, wherein said firstregion is nucleic acid.
 13. The probe of claim 11, wherein said probeconsists of 15 to 100 optionally modified nucleosides and one or moreblocking groups located at the 3′ terminus of said probe, wherein eachof said optionally modified nucleosides independently has, a purine orpyrimidine moiety independently selected from the group consisting ofinosine, uracil, adenine, guanine, thymine and cytosine; and a sugarmoiety independently selected from the group consisting of deoxyribose,2′-methoxy ribose, and ribose; and each of said optionally modifiednucleosides is joined together by an internucleoside linkageindependently selected from the group consisting of phosphodiester,phosphorothioate, and methylphosphonate.
 14. The probe of claim 13,wherein at least 80% of said optionally modified nucleosides has apurine or pyrimidine moiety independently selected from the groupconsisting of adenine, guanine, thymine and cytosine, and a deoxyribosesugar moiety; and at least 80% of said internucleoside linkages joiningsaid optionally modified nucleosides are phosphodiester.
 15. The probeof claim 14, wherein said probe consists of 35 to 70 independentlyselected nucleotides and said one or more blocking groups.
 16. The probeof claim 13, wherein said one or more blocking groups are selected fromthe group consisting of phosphorothioate, alkane-diol residue,cordycepin, and an alkyl group.
 17. The probe of claim 16, wherein saidfirst region has a nucleotide base sequence similarity of at least 75%with at least one of SEQ ID Nos.1, 2, 3, 4, 5 and
 6. 18. The probe ofclaim 17, wherein said first region has a sequence similarity of 75% to95% with SEQ ID NO:
 3. 19. The probe of claim 1, wherein said probecontains a region of self-complementarity.
 20. The probe of claim 11,wherein said probe contains a region of self-complementarity.
 21. Theprobe of claim 1, wherein said second region is present and the 3′ endof said second region is joined to the 3′ end of said first region by anon-nucleotide linker.
 22. The probe of claim 11, wherein said secondregion is present and the 3′ end of said second region is joined to the3′ end of said first region by a non-nucleotide linker.
 23. The probe ofclaim 1, wherein said first region cannot be used to produce saidfunctional double-stranded promoter sequence.
 24. The probe of claim 11,wherein said first region cannot be used to produce said functionaldouble-stranded promoter sequence.